Lung diseases, including inherited disorders such as Cystic Fibrosis (CF) and Surfactant Protein B (SP-B) Deficiency remain an issue in pediatric populations. SP-B deficiency is a rare lung disease where protein and fat molecules accumulate in the distant parts of the lungs and affect breathing. The disease is caused by a deficiency of the lung surfactant protein B, primarily due to a defect in the SFTPB gene which encodes the pulmonary-associated surfactant B protein (SPB), an amphipathic surfactant protein essential for lung function and homeostasis after birth. The most common mutation in SP-B deficiency is a mutation designated “121ins2” which results in the nucleotide “C” at position 131 being converted into “GAA.”
CF is an autosomal recessive disorder affecting 1 in 1500 to 4000 live births, and is one of the most common inherited pediatric disorders. The primary defect in CF is in the regulation of epithelial chloride transport by a chloride channel protein encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. See, e.g., Kerem et al. (1989) Science 245:1073-1080; Kreda et al. (2005) Mol Biol Cell 16:2154-2167. About 70% of mutations observed in CF patients result from deletion of three base pairs in CFTR's nucleotide sequence, resulting in the loss of the amino acid phenylalanine located at position 508 in the protein (a mutation referred to as ΔF508). In a wild type genome, amino acid 507 is an isoleucine, and is encoded by the codon TAG where the G is nucleotide 1652 in the gene. Amino acid 508 is a phenylalanine, encoded by AAA. In the Δ508 mutation, the G from the 507 codon is deleted along with the first two As of the 508 codon, such that the mutation has the sequence TAA at the deleted 507-508 encoding position. TAA also encodes an isoleucine, but the phenylalanine at wild type position 508 is lost. For the ΔI507 deletion, either the isoleucine at position 506 or 507 is deleted. For this mutation, the nucleotides at 1648-1650 or 1651-1653 are lost, or some combination thereof to result in only one isoleucine in the resultant protein. Compound (heterozygous) mutations (ΔF508 and ΔI507) have also been documented. See, e.g., Orozco et al. (1994) Am J Med Genet. 51(2):137-9. CF patients, either compound heterozygous ΔI507/ΔF508 or homozygous ΔF508/ΔF508, fail to express the fully glycosylated CFTR protein and the partially glycosylated protein is not expressed on the cell surface (see, e.g., Kreda et al. (2005) Mol Biol Cell 16:2154-2167; Cheng et al. (1990) Cell 63:827-834) as is required for CFTR function. Individuals bearing either the ΔI507 or ΔF508 CFTR mutations at only one allele (i.e. wt/ΔI507 or wt/ΔF508) are CF carriers and exhibit no defects in lung cell function. See, e.g., Kerem et al. (1990) Proc Natl Acad Sci USA 87:8447-8451.
Although several organ systems are affected by mutations in the CFTR gene, recurrent pulmonary infections are responsible for 80 to 90% of the deaths in CF patients. There is some controversy as to which human lung cell types express CFTR, although recent data indicate that CFTR expression is greatest in the proximal lung, and is predominantly expressed by ciliated cells present in surface airway epithelium. Kreda et al. (2005) Mol Biol Cell 16:2154-2167; Engelhardt et al. (1992) Nat Genet 2:240-248; Engelhardt et al. (1994) J Clin Invest 93:737-749.
Attempts to treat CF via in vivo gene therapy have been hindered by the immunogenic recognition and clearance of the viral vector used to deliver the CFTR transgene, failure to detect long-term expression of CFTR, and likely an inability to achieve stable transduction of relevant stem/progenitor cell populations in the lung Mueller & Flotte (2008) Clin Rev Allergy Immunol 35:164-178; Anson et al. (2006) Curr Gene Ther 6:161-179. Recently there have been reports of the isolation of human lung stem cells (see Kajstura et al., (2011) New England Journal of Medicine 364(19):1795). The authors report that these cells could be isolated, maintained in culture and re-introduced into damaged mouse lungs in vivo, where they were able to structurally integrate into the tissue and reform bronchioles, alveoli and pulmonary vessels.
Thus, there remains a need for the development of novel anti-CF strategies, including treatments and model systems (in vitro such as cell lines and in vivo animal systems) to model and treat CF based on investigation of CFTR mutations and develop stem cells for transplantation and treatment of pulmonary diseases.